Serological Profile of SARS-CoV-2 in Unvaccinated Blood Donors: A Nationwide Study During the Fifth Wave of the Pandemic in Iran

Background: Serological studies can demonstrate pathogen circulation in regional populations and reflect public health mea-sures' effectiveness during different pandemic phases. By late November 2021, coinciding with the third pandemic wave, the sero-prevalence of SARS-CoV-2 spike IgG antibodies among the Iranian population was 32.63%. Objective(s): This study aimed to assess the Iranian population's seroprevalence during the fifth pandemic wave by analyzing donated blood samples. Method(s): This population-based cross-sectional study was conducted on Iranian blood donors referred to all 31 main provincial capitals between August 2021 and September 2021. The participants selected through quota sampling were asked to complete a questionnaire on socio-demographics and coronavirus disease 2019 (COVID-19)-related information. Also, SARS-CoV-2 spike IgG antibodies were measured in serum samples using SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) kits. The seroprevalence was weighted based on the gender and age groups of the population and then adjusted for test performance. Result(s): Totally 3,339 blood donors participated in this study. The overall population-weighted seroprevalence adjusted for test performance was 52.67% (95% confidence interval (CI): 50.14-55.21). Seroprevalence was higher among participants with a high school diploma (55.45%, 95% CI 50.61-60.29), a positive history of close contact with COVID-19 patients (65.23%, 95% CI 60.83-69.63), and previous positive COVID-19 PCR tests (86.51%, 95% CI 82.32-90.7). Conclusion(s): More than half of the study population was exposed to SARS-CoV-2, indicating a 1.7-fold increase in the seroprevalence between late November 2020 and mid-September 2021. Our finding illuminated the pattern of Iran's fifth wave of the pandemic.Copyright © 2023, Author(s).

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

The effect of circulating cytokines on cardiovascular patients infected with the Coronavirus

The coronavirus illness (COVID-19) is caused by serious acute respiratory disorder coronavirus 2 (SARS-CoV-2), moreover known as the COVID-19 virus. After the first-ever reports of COVID-19 in December 2019, the malady spread quickly. In January 2020, the WHO announced the outbreak a Public Health Emergency of Worldwide Concern, and by March 2020, the WHO characterized the episode as a global widespread . The current study aimed to detect the effect of SARS-CoV-2 infection in heart patients and study their immune response by detecting the levels of some cytokines, which may end in a cytokine storm and may lead to death. In this study, one hundred-eight subjects were enrolled on two comparison case-control groups, the case group included 54 patients suffering from SARS-COV2, all were selected from those who were admitted to the Intensive Care Unit (ICU), and were diagnosed by a specialist physician with severe acute respiratory syndrome due to SARS-COV2 documented by Real-Time Polymerase Chain Reaction( RT-PCR ) besides other clinical and laboratory criteria in Marjan Medical City in Babylon province, AL-Amal Hospital for Communicable Diseases and AL-Hakeem Hospital, Najaf/Iraq, for a period from March 2022 to October 2022 to evaluate the role of some selected serological among patients with SARA-COV2 . The control group in this study included 54 subjects, divided into three groups (Apparent Healthy, patients suffered from SARS-COV2, patients suffered from CVD). Blood samples were examined through immunological methods, and an enzyme-linked immunosorbent assay (ELISA) was adopted for the detection of the concentration of TNF-alpha, IL6, IL-10,1L-12 and CCL2 .The immunological evaluation to clarify the theory of cytokines storm carried in the present study revealed that (TNF-alpha, IL6, IL-10,1L-12, and CCL2) for patients with COVID-19 and CVD was significantly higher than all the comparison group. The study reported that interleukin (6, 10, 12) and TNF-a are significantly increased in patients with covid19, CVD, and COVID-19 patients only, compared to healthy people. furthermore, IL-6 and IL-12 levels increased in patients with CVD only when compared to healthy people. There is a significant increase in CCL2 in all study groups compared to healthy people who have lower levels and this study indicated that the infection with Covid disease was severe and critical in most patients with CVD. This increased the number of deaths among them.Copyright © 2021 Muslim OT et al.

A cross-sectional serosurvey of SARS-CoV-2 and co-infections in stray cats from the second wave to the sixth wave of COVID-19 outbreaks in Spain.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. Among domestic animals, cats are more susceptible to SARS-CoV-2 than dogs. The detection of anti-SARS-CoV-2 antibodies in seemingly healthy cats and/or infected cats which are in close contact with infected humans has been described. The presence of animals that tested positive by serology or molecular techniques could represent a potential transmission pathway of SARS-CoV-2 that can spill over into urban wildlife. This study analyses the seroprevalence variation of SARS-CoV-2 in stray cats from different waves of outbreaks in a geographical area where previous seroepidemiological information of SARS-CoV-2 was available and investigate if SARS-CoV-2-seropositive cats were exposed to other co-infections causing an immunosuppressive status and/or a chronic disease that could lead to a SARS-CoV-2 susceptibility. For this purpose, a total of 254 stray cats from Zaragoza (Spain) were included. This analysis was carried out by the enzyme-linked immunosorbent assay using the receptor binding domain of Spike antigen and confirmed by serum virus neutralization assay. The presence of co-infections including Toxoplasma gondii, Leishmania infantum, Dirofilaria immitis, feline calicivirus, feline herpesvirus type 1, feline leukemia virus and feline immunodeficiency virus, was evaluated using different serological methods. A seropositivity of 1.57% was observed for SARS-CoV-2 including the presence of neutralizing antibodies in three cats. None of the seropositive to SARS-CoV-2 cats were positive to feline coronavirus, however, four SARS-CoV-2-seropositive cats were also seropositive to other pathogens such as L. infantum, D. immitis and FIV (n = 1), L. infantum and D. immitis (n = 1) and L. infantum alone (n = 1).Considering other pathogens, a seroprevalence of 16.54% was detected for L. infantum, 30.31% for D. immitis, 13.78%, for T. gondii, 83.86% for feline calicivirus, 42.52% for feline herpesvirus type 1, 3.15% for FeLV and 7.87% for FIV.Our findings suggest that the epidemiological role of stray cats in SARS-CoV-2 transmission is scarce, and there is no increase in seropositivity during the different waves of COVID-19 outbreaks in this group of animals. Further epidemiological surveillances are necessary to determine the risk that other animals might possess even though stray cats do not seem to play a role in transmission.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01-10.0 and 50-300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV-vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.

Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals.

As there are limited data on B cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine.Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n=30), WT infected (n=30), delta infected (n=30), omicron infected+vaccinated (n=20) and Sinopharm (BBIBP-CorV) vaccinees (n=30). We then investigated the sensitivity and specificity of these immunodominant regions and analysed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274 to 297 and aa 365 to 388, were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

A linear B-cell epitope close to the furin cleavage site within the S1 domain of SARS-CoV-2 Spike protein discriminates the humoral immune response of nucleic acid- and protein-based vaccine cohorts.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

A serological survey of pathogens associated with the respiratory and digestive system in the Polish European bison (Bison bonasus) population in 2017-2022.

BACKGROUND: The European bison (Bison bonasus) is a near threatened species and requires health monitoring. The aim of the present study was to determine the prevalence of antibodies to pathogens known to cause respiratory and digestive illness in ruminants. RESULTS: In the studied 328 European bison, the highest seroprevalence was observed for Bovine herpesvirus-1 (BoHV-1) (50.27%), Bovine Coronavirus (BCoV) (26.36%), and Bluetongue Virus (BTV) (12.83%). For Mycoplasma bovis strains and Bovine Viral Diarrhea Virus (BVDV), positive results were rare. Interestingly, a higher prevalence of BTV antibodies was noted in the northeastern populations and older animals. CONCLUSIONS: Our findings indicate that the Polish European bison population appears to have considerable contact with BoHV-1; however, this does not appear to be of great significance, as clinical symptoms and post-mortem lesions are rarely noted in Polish European bison population. The high seroprevalence of BTV in the north-east of Poland is an ongoing trend, also noted in previous studies. It is possible that European bison may perpetuate the virus in this region. This is the first report of antibodies for BCoV in European bison.

A Customized Novel Blocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered emerging alphacoronavirus. SADS-CoV shares over 90% genome sequence identity with bat alphacoronavirus HKU2. SADS-CoV was associated with severe diarrhea and high mortality rates in piglets. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. Here, monoclonal antibody (MAb) 6E8 against SADS-CoV N protein accurately recognized SADS-CoV infection. Then, MAb 6E8 was utilized as a blocking antibody to develop blocking ELISA (bELISA). We customized the rN coating antigen with concentration 0.25 µg/mL. According to receiver operator characteristic curve analysis, the cutoff value of the bELISA was determined as 38.19% when the max Youden index was 0.955, and specificity was 100%, and sensitivity was 95.5%. Specificity testing showed that there was no cross-reactivity with other serum positive swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine sapelovirus (PSV). In conclusion, we customized a novel and high-quality blocking ELISA for detection of SADS-CoV infection, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection. IMPORTANCE SADS-CoV was reported to be of high potential for dissemination among various of host species. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. We customed a novel and high-quality bELISA assay for detection of SADS-CoV N protein antibodies, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection.

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions to PEG-containing Covid-19 vaccines: Evidence for immunogenicity of PEG.

A small fraction of recipients who receive polyethylene-glycol (PEG)-containing COVID-19 mRNA-LNP vaccines (Comirnaty and Spikevax) develop hypersensitivity reactions (HSRs) or anaphylaxis. A causal role of anti-PEG antibodies (Abs) has been proposed, but not yet been proven in humans.We used ELISA for serial measurements of SARS-CoV-2 neutralizing Ab (anti-S) and anti-PEG IgG/IgM Ab levels before and after the first and subsequent booster vaccinations with mRNA-LNP vaccines in a total of 291 blood donors. The HSRs in 15 subjects were graded and correlated with anti-PEG IgG/IgM, just as the anti-S and anti-PEG Ab levels with each other. The impacts of gender, allergy, mastocytosis and use of cosmetics were also analyzed. Serial testing of two or more plasma samples showed substantial individual variation of anti-S Ab levels after repeated vaccinations, just as the levels of anti-PEG IgG and IgM, which were over baseline in 98-99 % of unvaccinated individuals. About 3-4 % of subjects in the strongly left-skewed distribution had 15-45-fold higher values than the median, referred to as anti-PEG Ab supercarriers. Both vaccines caused significant rises of anti-PEG IgG/IgM with >10-fold rises in about âˆ¼10 % of Comirnaty, and all Spikevax recipients. The anti-PEG IgG and/or IgM levels in the 15 vaccine reactors (3 anaphylaxis) were significantly higher compared to nonreactors. Serial testing of plasma showed significant correlation between the booster injection-induced rises of anti-S and anti-PEG IgGs, suggesting coupled anti-S and anti-PEG immunogenicity.Conclusions: The small percentage of people who have extremelevels of anti-PEG Ab in their blood may be at increased risk for HSRs/anaphylaxis to PEGylated vaccines and other PEGylated injectables. This risk might be further increased by the anti-PEG immunogenicity of these vaccines. Screening for anti-PEG Ab "supercarriers" may help predicting reactors and thus preventing these adverse phenomena.

Determination of antibodies to SARS-CoV-2 in patients with coronavirus infection

COVID-19, caused by the novel SARS-CoV-2 virus, poses major challenges for global public health. The detection of antibodies in blood serum is one of the important methods for diagnostics of COVID-19 patients. The main aim was to study the dynamics of the appearance of neutralizing antibodies and antibodies to the SARS-CoV-2 proteins in COVID-19 patients sera. Material and methods. The blood sera of four groups of people were studied: "intact" donors (blood sera were collected in 2016-2019);patients with a laboratory-confirmed diagnosis of acute respiratory viral infection;patients with influenza (antibodies to the influenza virus have been identified) and patients with a PCR confirmed diagnosis of COVID-19. Blood sera were analyzed in ELISA with commercial kits for detection of IgG to SARS-CoV-2 (N, S) proteins and total antibodies to RBD of protein S and in neutralization test (NT). Results and discussion. Antibodies to SARS-CoV-2 were not detected in paired blood sera of people from groups 1-3 by ELISA and NT. At the time of hospitalization of patients with COVID-19 in the sera of 12 (19%) patients antibodies to SARS-CoV-2 were absent when they were determined by NT and ELISA. In blood sera taken 4-9 days after hospitalization, neutralizing antibodies and antibodies to at least one viral protein were detected in ELISA. Conclusion. At the time of hospitalization, the overwhelming majority of patients had a humoral immune response to the SARS-CoV-2. In the dynamics of observation, the levels of antibodies to SARS-CoV-2 proteins increased, to a greater extent to RBD.Copyright © 2022 Geotar Media Publishing Group

Cuban Vaccines Abdala and Mambisa against Covid-19

Intro: With the first case of COVID-19 in Cuba on March 11, 2020, the Center for Genetic Engineering and Biotechnology in Havana began an extensive vaccine program. Two vaccines based on RBD recombinant protein were developed, one for systemic administration "Abdala" and one mucosal vaccine "Mambisa". Abdala received the EUA in July 2021 and "Mambisa" completed its clinical development as a booster dose for convalescent subjects. Method(s): Two doses (25 and 50 microg) and two schedules (0-14-28 and 1-28-56 days) were evaluated in phase I clinical trials with volunteers 19 to 54 years old. The phase II and III clinical trials were also double-blind, randomized, and placebo-controlled, and included respectively 660 and 48,000 volunteers from 19 to 80 years. The anti-RBD titers were evaluated using a quantitative ELISA system developed at the Center for Immunoassay, Havana Cuba, and ELECSYS system from Roche. The RBD to ACE2 plate-based binding competitive ELISA was performed to determine the inhibitory activity of the anti-RBD polyclonal sera on the binding of the hFc-ACE2 coated plates. The neutralization antibody titers were detected by a traditional virus microneutralization assay (MN50). Finding(s): The Abdala vaccine reached 92.28% efficacy. The epidemic was frankly under control in Cuba after the vaccine introduction having reached the highest levels of cases and mortality in July 2021 with the dominance of the Delta strain. The peak of the Omicron wave, unlike other countries, did not reach half of the cases of the Delta wave with a significant reduction in mortality. The mucosal vaccine candidate "Mambisa" completed its clinical development as a booster dose for convalescent subjects reaching the trial end-point. Conclusion(s): Vaccine composition based on RBD recombinant antigen alone is sufficient to achieve high vaccine efficacy comparable to mRNA and live vaccine platforms. The vaccine also protects against different viral variants including Delta and Omicron strains.Copyright © 2023

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Seroprevalence of human brucellosis among the tribal and non-tribal population residing in an eastern state of India: Findings from the state-wide serosurvey.

Background: Brucellosis is a neglected zoonotic disease found predominantly in lower- and middle-income countries (LMICs), causing significant public health concern in India. The objective of this study was to assess the prevalence of human brucellosis in Odisha, India among community members involved in animal husbandry as a common practice. Method: This cross-sectional study included 817 adult participants from 11 districts in Odisha. Four districts from the Northern division, four districts from the Central division, and three districts from the Southern division were selected for the study. Blood samples were collected during a COVID-19 serosurvey in Odisha conducted from 1st to 17th September 2021. Immunoglobulin-G (IgG) antibodies were measured against Brucella using a commercial ELISA kit. Point estimates at 95% confidence intervals (CIs) and adjusted odds ratio were calculated. Results: The overall prevalence of anti-Brucella IgG antibodies was calculated at 16.65% (95% CI: 14.19-19.42). The highest seropositivity was found in Sambalpur district (29.73%; 95% CI: 16.43-47.16) and the lowest was determined in Mayurbhanj district (4.44%; 95% CI: 0.99-15.60). Compared to males, females were more prone to contracting the disease (AOR: 1.13; 95% CI: 1.05-1.67). Participants from rural settings had higher prevalence of anti-Brucella IgG antibodies than urban dwellers (AOR: 4.53; 95% CI: 1.73-11.86). Conclusion: This study revealed that human brucellosis was associated with sociodemographic factors like gender, living settings, and household numbers. To prevent brucellosis, screening should be initiated, infected humans should be treated early, and the public should be educated about risk factors and preventive measures.

Detection of Influenza D-Specific Antibodies in Bulk Tank Milk from Swedish Dairy Farms.

Influenza D virus (IDV) has been detected in bovine respiratory disease (BRD) outbreaks, and experimental studies demonstrated this virus's capacity to cause lesions in the respiratory tract. In addition, IDV-specific antibodies were detected in human sera, which indicated that this virus plays a potential zoonotic role. The present study aimed to extend our knowledge about the epidemiologic situation of IDV in Swedish dairy farms, using bulk tank milk (BTM) samples for the detection of IDV antibodies. A total of 461 and 338 BTM samples collected during 2019 and 2020, respectively, were analyzed with an in-house indirect ELISA. In total, 147 (32%) and 135 (40%) samples were IDV-antibody-positive in 2019 and 2020, respectively. Overall, 2/125 (2%), 11/157 (7%) and 269/517 (52%) of the samples were IDV-antibody-positive in the northern, middle and southern regions of Sweden. The highest proportion of positive samples was repeatedly detected in the south, in the county of Halland, which is one of the counties with the highest cattle density in the country. In order to understand the epidemiology of IDV, further research in different cattle populations and in humans is required.

Ace2 Substrate Cleavage Effect Associated with Covid-19 Patient Plasma Immunoglobulin

Background: Many mechanisms responsible for COVID-19 pathogenesis are well-known, but COVID-19 includes features with unclear pathogenesis, like autonomic dysregulation, coagulopathies, and high levels of inflammation. The SARS-CoV-2 spike protein receptor binding domain (RBD) receptor is angiotensin converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop immunoglobulins (Igs) that have negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity - ACE2-like 'abzymes'. Method(s): To explore this hypothesis, we studied 67 patients hospitalized with COVID-19 who had disodium ethylenediaminetetraacetate (EDTA) anticoagulated plasma samples available, obtained about 7 days after admission. We used commercially available fluorometric ACE2 assays (Abcam), and a SpectraMax M5 microplate reader (Molecular Devices), measuring Relative Fluorescent Unit (RFU, Ex/Em = 320/420 nm;RFU) in a kinetic mode every 20 min at 37C. ACE2 inhibitor provided in the assay kit was used for additional controls. In some control experiments, we added Zn2+ to plasma, or conducted serial dilutions to decrease Zn2+. To deplete Igs, we passed plasma samples through a 0.45 mum filter to remove large particles, then passed the material through 100kDa cut-off ultrafiltration membrane (PierceTM) columns, and finally used protein A/G Magnetic Beads (Life Technologies) to specifically deplete Ig, removing >99.99% of Ig as assessed with a human IgG ELISA Kit (Abcam). Result(s): ACE2 is a metalloprotease that requires Zn2+ for activity. However, we found that the plasma of 11 of the 67 patients could cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using EDTA anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+ was added, or the plasma was diluted to decrease EDTA concentration. After processing samples by size exclusion and protein A/G adsorption, the plasma samples did not cleave the ACE2 substrate peptide. Conclusion(s): The data suggest that some patients with COVID-19 develop Igs with activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure features of COVID-19 pathogenesis. (Figure Presented).

Investigation of TNF-alpha and IL-4 in Pfizer/BioNTech vaccinated people: A comparative study

Background: The necessity for a vaccine to prevent this disease has been made abundantly clear by the appearance of the new SARS-CoV-2 and COVID-19. The most reliable method of halting the spread of infectious illnesses is vaccination. Since they were first made available to the general public more than 200 years ago, vaccines have saved millions of lives. Method(s): There were eighty-one (81) participants in total in the study. Individuals ranged in age from 18 to 66 and had recently received COVID-19 mRNA Pfizer/BioNTech [BNT162b2] vaccination injections. They were given two injections of the vaccine of 30 g and 0.3 mL, twenty-one (21) days apart. Before the first vaccination, blood samples were collected. This procedure was repeated on days 7-10 after the first vaccination, and on days 7-10 after the second dose. All samples were tested for IL-4, and TNF-alpha using a High Sensitivity Human ELISA Kit corresponding to each marker (Elabscience/United State). Result(s): There was no significant increase in IL-4 levels in all groups, TNF-alpha results showed increased after the first and second doses compared to before vaccination, and the increase after the second dose is greater than the first dose. Conclusion(s): Our research demonstrated that vaccinations caused Th1 biases and prevented Th2 responses in all groups.Copyright © 2023, Codon Publications. All rights reserved.

Comparative ELISA Detection of SARS-COV-2 Monoclonal Antibodies in Patients' Serum, Saliva, and Nasal Fluid in Iraq

Precise management of COVID-19 infection and global pandemic requires a precise assay of SARS-COV-2 antibodies. Spike protein and RBD specific serum monoclonal antibodies has been known to be elicited with the currently approved mRNA vaccine for COVID-19. Since combating SARS-CoV-2 infection begins as early at the viral entry port, mediated by mucosal immunity;whether the currently approved vaccines have the capacity to elicit mucosal immunity is still to be studied. Method;We conducted our research to detect SARS-Cov-2 IgA-RBD and IgG-NCP, in a set of a randomized three patients' groups (total 55 subjects, randomized into 20 (Non previously infected control, 20 fully vaccinated people from AlSalam teaching hospital vaccination ward upon receiving the vaccine doses, and 15 patients newly recovered from SARS-CoV-2 infection from AlShifaa quarantine hospital after remission from COVID-19 infection and preparing to discharge) in three fluid samples serum, saliva and nasal fluid, for a total of three samples per participant, to make a total of 165 samples to be tested, using a commercial immunoassay kit (Anti-SARS-CoV-2 ELISA RBD IgA, and Anti-SARS- CoV-2 NCP ELISA IgG) the study was designed to assess the immunoglobulins levels in each sample from different health status and anatomical locations to give the statistical difference and the correct conclusion regarding this study. Statistical analysis was performed using SPSS version 24, as well as Microsoft excel version (16.0.15028), test equations were performed via Mann-Whitney U Test, as well as The Kruskal-Wallis H test, to evaluate the non-gaussian distribution among the study group, for each of the outcomes, p < 0.05 was undertaken as statistically significant. Results;Our work showed that two doses of mRNA BNT162b2 vaccine Comirnaty (Pfizer/BioNTech, New York, NY, USA), elicited a robust anti SARS-CoV-2 nasal and salivary IgA and IgG monoclonal antibody protection (as compared to control 0.625 COI, the vaccinated subjects resulted in a higher median salivary concentration of IgG NCP values as 1.69 COI, with a p-value of 0.0001. same goes for median salivary concentration of IgA RBD with value of 2.875 ± 1.276, as compared to control median values of 0.667 ± 0.208), (as compared to control 0.462 ± 0.2369, the vaccinated subjects resulted in a higher median nasal concentration of IgG NCP values as 1.944 ± 0.694, with a p-value of 0.0001. Same goes for median nasal concentration of IgA RBD with value of 1.941 ± 0.53, as compared to control median values of 0.681 ± 0.226). Two injections 25 days a part shown to trigger a stronger titer of protective immunity as compared to single early shot, still it's less robust compared to the titers measured for the recovered subjects from COVID-19 infection. Taking into consideration that there was a lack of trustable ELISA based assays with specially designed kit for this purpose, focusing on nasal and salivary secretions, the current study that our pre investigational and investigational data are consistent. The results of this study indicates that a protective antigen specific nasal and salivary monoclonal antibody, can be triggered following proper vaccination regimen with mRNA COVID-19 vaccine, so this paves the way for using mucosal protective antibodies detection kits, as a suitable alternative option as a less invasive method compared to serum investigations, for detecting the protection against SARS-Cov-2 infection, induced by vaccine. The vaccinated subjects are significantly better tested via IgA in Saliva as compared to IgG, because of the higher median values for the IgA vs IgG, 2.875 ± 1.276 and 2.083 ± 1.610 respectively with a p-value of 0.008. in a similar state the IgA is the best test for the convalescent subjects in term of nasal fluid samples investigations, as the median value are comparatively higher than median value of IgG, 2.024 ± 0.520 and 1.786 ± 1.115 respectively. © 2023 Israa University Journal of Applied Science. All rights reserved.

Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus

[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.